I. Field of the Invention
This invention relates to the culturing of somatic embryos, particularly those of gymnosperm plant species and especially conifers. More particularly, the invention relates to the culturing of somatic embryos in which abscisic acid (ABA) is employed for embryo development.
II. Description of the Prior Art
Somatic embryogenesis offers the potential to clonally produce large numbers of plants of many species at low cost. Somatic embryos develop without the surrounding nutritive tissues and protective seed coat found with zygotic embryos, so research has been directed to causing somatic embryos to functionally mimic seeds with regard to efficient storage and handling qualities. The development of techniques for somatic embryogenesis in conifers has greatly improved the ability to culture conifer tissues in vitro and now offers the means to clonally propagate commercially valuable conifers. However, it is necessary to further reduce production costs to make somatic embryogenesis affordable to industry. Thus, there is a need in the technology for improvement of the efficiency of embryo production and in the quality and vigor of plants resulting from somatic embryos from all species of conifers.
Somatic embryogenesis in plants is a multistage process consisting of induction, proliferation, maturation (embryo development), and germination, that requires specific culture conditions, including nutrient media compositions provided for each step of the multistep process. Thus, in general for most conifers, an auxin and cytokinin and a low osmoticum are required in media for induction and proliferation of embryogenic tissues. For further embryo development it is often beneficial to increase the osmotic concentration, and to replace the auxin and cytokinin with ABA.
Conifer somatic embryos appear different to somatic embryos of monocotyledonous and dicotyledonous species in that ABA should be supplied as early as possible in maturation protocols in order to promote embryo maturation. Merely reducing or eliminating auxin and cytokinin levels, as has been successful for maturation of somatic embryos of many angiosperm species (Ammirato 1983, Handbook of Plant Cell Culture, Vol. 1, pp. 82-123) led to infrequent or poor maturation in conifer embryos and more often resulted in browning and death of the immature somatic embryos. Furthermore, it appears that ABA should be applied for longer periods and at higher levels than generally applied to angiosperm somatic embryos.
There has been a trend for using increasingly higher concentrations of ABA to promote the maturation of conifer somatic embryos. This trend probably results from a need to inhibit precocious germination late in maturation which has become more apparent following the increasingly longer maturation times being used. Thus, ABA was first successfully used by Hakman and von Arnold 1988 (Physiol. Plant. 72:579-587) and von Arnold and Hakman 1988 (J. Plant Physiol. 132:164-169), at 7.6 μM. Dunstan et al. 1988 (Plant Sci. 58:77-84) subsequently found 12 μM ABA to be better. Shortly after, Attree et al. 1990 (Can. J. Bot. 68:2583-2589) reported that 16 μM was optimal. Roberts et al. 1990 (Physiologia Plantarum 78; 355-360) have shown that for some species of spruce, ABA at 30-40 μM could be used to promote maturation and yield mature embryos with storage protein polypeptides comparable to zygotic embryos. Such high levels were necessary to prevent precocious germination and allow maturation to proceed to later stages. Dunstan et al. 1991 (Plant Sci. 76:219-228) similarly found that high levels could permit embryo maturation. Unfortunately, high ABA levels used throughout the development period also increased the frequency of developmentally abnormal embryos. Subsequently, much higher ABA concentrations have been described. Becwar et al., U.S. Pat. No. 5,506,136 described ABA in development media at up to 120 μM. Dunstan et al., 1997 (Journal of Expt. Bot. 48, 277-287) suggested that a remedy to prevent precocious germination of conifer somatic embryos is to transfer cultures to fresh medium with ABA in the maturation culture period, as is commonly done. It is stated that exposure to fresh ABA is unlikely to lead to greatly improved yields of mature somatic embryos, unless the population of immature embryos remains sizable, but is more likely to lead to improvement in the quality of the mature somatic embryos through deposition of storage product and prevention of precocious germination. Dunstan et al. 1997 (J. Plant Physiol.) showed that the availability of ABA decreases during culture so can lead to precocious germination. They suggested that this is generally attributed to a low starting concentration of ABA, and the authors also suggest that extending the use of ABA during the maturation phase by periodic transfer to fresh nutrient medium will extend ABA availability. Uddin 1993 (U.S. Pat. No. 5,187,092) describes using various combinations and proportions of glucose, maltose, ABA and/or indolebutyric acid to promote maturation of conifer somatic embryos. This patent suggests that conifer somatic embryos should be cultured in the presence of at least 3% maltose and at least 10 μM ABA.
Attree et al (U.S. Pat. No. 5,464,769) described the combined use of a water stress and ABA during the embryo maturation process to stimulate maturation frequencies and promote further maturation of the embryos, and to increase dry weight and lower moisture content, leading to desiccation tolerance. Constant levels of ABA were maintained during development of the embryos. With regard to the non-plasmolysing water stress, a non-plasmolysing high molecular weight compound such as PEG, however, other non-plasmolysing water stresses such as environmental stresses or increased gelling agent were also suggested, and increased gelling agent was also described in U.S. Pat. No. 6,200,809.
Kapik et al. 1995 (Tree Physiology 15, 485-490), and Kong et al., 1997 (Physiologia Plantarum 101, 23-30) showed that endogenous ABA rises during seed and zygotic embryo development then falls during late development. Therefore, it was suggested that ABA should be moderately high at the start of development then decreased throughout development to low levels or to zero at the end of the culture period, so promoting germination.
Thus, in U.S. Pat. No. 5,034,326 Pullman et al. (1991) describe a method for developing tissue culture induced coniferous somatic embryos into well-developed cotyledonary embryos. The method comprises a multi-stage culturing process in which early stage embryos are cultured on a late stage medium comprising a significantly higher osmotic potential along with moderately high ABA and an absorbent material to gradually reduce the level of available ABA over time. A critical aspect of this method lies in the inclusion of the absorbent material in the embryo development medium. Absorbent materials suggested include activated charcoal and silicates. The absorbent is used to slowly reduce the ABA and remove metabolic waste products. The purpose of this reduction in ABA was to follow the natural tendency in embryo development. It was suggested that as development approaches completion, the presence of lesser amounts of ABA is required. Similarly Gupta et al. (1991) in U.S. Pat. No. 5,036,007 describe a similar method. In Douglas fir culture ABA is reduced from about 10-20 μM at the start of development to less than about 3 μM or even to zero.
A similar method was described in U.S. Pat. No. 5,236,841, by Gupta et al. (1993), however, the invention relates to the use of gradually decreasing amounts of the plant hormone abscisic acid during the time when the embryos are further developed into cotyledonary embryos by stepwise subcultures. It was suggested that when transfers to fresh medium are made that the initial ABA level of the fresh medium should not be higher than the final level of the medium at the end of the preceding culture period. More recently, however, Gupta et al. (1996) in U.S. Pat. No. 5,482,857 found that when using activated charcoal ABA was not necessary for cotyledonary embryo development of Douglas fir.
U.S. Pat. No. 6,627,441 describes a method for producing viable mature conifer somatic embryos consisting of water stressing the somatic embryos in medium containing ABA that is increasing during development including towards the mid point of cotyledonary development when the tendency for precocious germination is the highest prior to water contents becoming sufficiently low. Also envisaged is a method where the ABA is rising progressively throughout development.
It has been suggested to use abscisic acid (ABA) or osmoticum for enhancing storage levels in plant cells. For example, it was shown that somatic embryos of Theobroma cacao could be induced to accumulate fatty acids approaching the composition of commercial cocoa butter by using a high sucrose concentration in the culture medium (Pence et al. 1981; Physiol. Plant. 53:378-384). Modifying the culture conditions by osmoticum concentration and/or ABA content similarly improved lipid accumulation in Brassica napus L. somatic (Avjioglu and Knox 1989; Ann. Bot. 63:409-420) and microspore (Taylor et al. 1990; Planta 181: 18-26) derived embryos as well as somatic embryos of carrot (Dutta and Appelqvist 1989; Plant Sci. 64: 167-177) and celery. Also, the level of storage lipids in P. abies somatic embryos was improved by optimising the ABA level to between 10-20 μm, but the somatic embryos contained about 4% of the lipid level obtained by zygotic embryos (Feirer et al. 1989; Plant Cell Rep. 8:207-209).
Embryo drying occurs naturally in most seeds, and has a role to play in the developmental transition between maturation and germination. Thus, desiccation led to enhanced germination of both zygotic and somatic embryos. Desiccation of whole somatic embryos is also an alternative method of germplasm storage. Somatic embryos produced continuously year-round could therefore be dried and stored until the appropriate planting season, or shipped to new locations. Conifer embryos treated with ABA and water stress can survive desiccation to low moisture contents (U.S. Pat. Nos. 5,464,769, 5,985,667, 6,340,594, 6,372,496).
ABA has also been used to in induction and maintenance media to promote induction and proliferation of conifer somatic embryos (U.S. Pat. Nos. 5,677,185, 5,856,191 and U.S. patent application 2002/0192818).
All of the above examples involving embryo tissue culture used synthetically produced and commercially available racemic ABA which consisted of 50% mixtures of (+)-ABA (S-ABA) and (−)-ABA (R-ABA). (−)-ABA is not biologically inert—it has hormonal activity in many bioassays, and is degraded by a different route in plant tissues from (+)-ABA—and at a substantially different rate (usually much slower). The effect of pure S(+)-ABA on conifer somatic embryogenesis is largely unknown. Dunstan et al. 1992 did compare a pure sample of S(+)-ABA with racemic on somatic embryos of spruce liquid suspension cultures and showed that the S(+)-ABA was metabolized completely into phaseic acid within seven days. The R(−)-ABA remained unchanged. The cultures were only treated for a maximum of eight days and no mature embryos were recovered and seedlings grown and no benefit was suggested for using S(+)-ABA over the racemic S,R(±)-ABA form.
The development of techniques for somatic embryogenesis in conifers has greatly improved the ability to culture conifer tissues in vitro and now offers the means to clonally propagate commercially valuable conifers. However, it is necessary to further reduce production costs to make somatic embryogenesis affordable to industry. Thus, there is a need in the technology for improvement of the efficiency of embryo production and of the quality and vigour of plants resulting from somatic embryos from all species of conifers.